Compositions for detecting ras gene proteins and cancer therapeutics

ABSTRACT

Compositions useful for detecting ras gene proteins are described consisting of GTP and a protein having an apparent reduced molecular weight of about 115,000-120,000 daltons, or fragments derived therefrom, that stimulate ras protein guanosine triphosphatase activity. Also described are methods whereby the compositions are used to identify cancer therapeutics.

FIELD OF THE INVENTION

This invention relates generally to the field of oncology, andparticularly to diagnostic compositions useful in testing for cancer.Additionally, the invention concerns compositions that can be employedboth as cancer diagnostics, as well as in a scheme for identifyingcancer therapeutics.

BACKGROUND OF THE INVENTION

Several genes have been identified that are thought to play a role inregulating normal cell growth. A subset of these genes, termed ras,consists of at least three members, N-ras, H-ras, and K-ras2. Alteredforms of ras, termed oncogenes, have been implicated as causative agentin cancer. Both the normal cellular genes, and the oncogenes encodechemically related proteins, generically referred to as p21.

Ras oncogenes, and their normal cellular counterparts, have been clonedand sequenced from a variety of species. Comparison of the structure ofthese two genes has revealed that they differ by point mutations thatalter the amino acid sequence of the p21 protein. Naturally occurringmutations in the ras oncogenes have been identified in codons 12, 13,59, and 61. In vitro mutagenesis work has shown that mutations in codon63, 116, and 119 also result in transforming activity. The mostfrequently observed mutation which converts a normal cellular ras geneinto its oncogenic counterpart is a substitution of gaycane at position12 by any other amino acid residue, with the exception of proline.Transforming activity is also observed if glycine is deleted, or ifamino acids are inserted between alanine at position 11 and glycine atposition 12.

Mutations at position 61 also play an important role in the generationof ras oncogenes. Substitution of glutamine for any other amino acid,except proline or glummac acid in the cellular ras gene yields rasoncogenes with transforming activity.

In relation to normal cellular ras genes and their oncogeniccounterparts, there are at least four known retrovital ras oncogeneswhich exhibit transforming activity. Unlike their non-retroviralanalogues, the retroviral genes exhibit two mutations. The biologicallysignificance of these double mutations is at present unclear.

Both the normal ras and oncogenic p21 proteins, regardless of theirphylogenetic origin, bind guanine nucleotides, GTP and GDP, and possessintrinsic GTPase activity. Temeles et al., Nature, 313:700 (1985). Thesignificance of these biochemical properties to the biologicalactivities of the ras proteins has been demonstrated as follows: first,microinjection of anti-ras antibodies that interfere with guaninenucleotide binding reverses the malignant phenotype of NIH 3T3 cellstransformed by ras oncogenes. Clark, et el., Proc. Natl. Acad. Sci.U.S.A., 82:5280 (1985); Feramisco, et al., Nature, 314:639 (1985).Second, ras oncogenic proteins that exhibit mutations which result inthe inability of p21 to bind guanine nucleotides do not transform NIH3T3 cells. Willumsen, et al., Mol. Cell. Biol., 6:2646 (1986). Third,some ras oncogenes produce p21 proteins that have much reduced GTPaseactivity compared to their normal cellular counterparts. The biologicalrole of GTPase activity associated with either ras or its oncogeniccounterpart remains unknown.

Recently a cytoplasmic factor has been identified which stimulatesnormal ras p21 GTPase activity, but does not effect GTPase activityassociated with the oncogenic routants. M. Trahey and F. McCormick,Science, 238:542 (1987). The activity has been associated with aprotein, termed GAP, which is the acronym for (GTPase activatingprotein. GAP is thought to be a cytoplasmic protein but is presumablycapable of moving from the cytosol to the plasma membrane where itinteracts with p21.

As alluded to above, ras oncogenes have been implicated in thedevelopment of a variety of tumors, and have been shown to be involvedin about 10-40% of the most common forms of human cancer. H. Varmus,Annual Rev. Genetics, 18:553 (1984); M. Barbacid, in Important Advancesin Oncology (1986), ed. B. DeVita, S. Helman, S. Rosenberg, pages 3-22,Philadelphia:Lippincott. For example, ras oneogenes have beenconsistenfly identified in carcinomas of the bladder, colon, kidney,liver, lung, ovary, pancreas and stomach. They also have been identifiedin hematopoietic tumors of lymphoid and myeloid lineage, as well as intumors of mesenchymal origin. Furthermore, melanomas, teratocarcinomas,neuroblastomas, and gliomas have also been shown to possess rasoncogenes.

Considering the possible association of ras oncogenes and cancer, therehas been considerable work focused on diagnostic tests for detecting thepresence of the oncogene product, p21, or the mutant oncogenes. Earlytests, which are still employed in many instances, identify the presenceof ras oncogenes in transfection assays which identify p21 by itsability to transform NIH 3T3 cells. Lane, et at., Proc. Natl., Acad.Sci. U.S.A., 78:5185 (1981); and B. Shilo, and R. A. Weinberg, Nature,289:607 (1981). This method is insensitive, laborious, and requires askilled laboratory technician to perform adequately.

A second diagnostic method centers around oligonucleotide probes toidentify single, point mutations in genomic DNA. This technique is basedon the observation that hybrids between oligonucleotides form a perfectmatch with genomic sequences, that is, nonmutated genomic sequences aremore stable than those that contain a single mismatch. The latter, ofcourse, being a point mutation in p21 associated with the ras oncogenes.Although this technique is clearly more sensitive and easier to performthan the transfection assay, it is nevertheless also cumbersome toperform. This is because there are theoretically almost 100 basesubstitutions which can yield ras oncogenes. Thus, in order to be ableto detect these substitutions multiple oligonucleotide probes must beemployed containing each of the three possible substitutions at aparticular residue. Bos, et al., Nature, 315:726 (1985); Valenzuela, etal., Nuc. Acid Res., 14:843 (1986).

In addition to the transfection and oligonucleotide assays, additionalnucleic acid hybridization techniques have been developed to identifyras oncogenes. One such method is based on the unusual electrophoreticmigration of DNA heteroduplexes containing single based mismatches indenaturing gradient gels. Myers et al., Nature, 313:495 (1985). Thistechnique only detects between about 25-40% of all possible basesubstitutions, and requires a skilled technician to prepare thedenaturing gradient gels. More sensitive techniques which arerefinements of this technique are described by Winter, et al., Proc.Natl., Acad. Sci. U.S.A., 82:7575 (1985); and Myers, et al., Science,230:1242 (1985).

Immunologic approaches have been taken to detect the product of the rasoncogenes. Antibodies, either polyclonal or monoclonal, have beengenerated against the intact ras oncogene p21, or against chemicallysynthesized peptides having sequences similar to oncogene p21, or thenon-transforming counterpart. U.S. patent application Ser. No. 938,581;EP Patent Publication 108,564 to Cline et al.; Tamura, et al., Cell,34:587 (1983); PCT Application WO/84/01389 to Weinberg et al. For themost part, unfortunately, antibodies have been disappointing asdiagnostic tools with which to identify ras oncogenic p21 in humantissue sections. This is because either the antibodies that have beengenerated to date recognize the normal cellular ras protein as well asthe oncogenic protein, or in those instances where a monocional antibodyhas been generated that specifically recognizes the oncogenic protein,it exhibits non-specific staining of tumor biopsies.

While ras oncogenic p21 is an effective tumorigenic agent, recentstudies have shown that normal ras p21 can induce the malignantphenotype. Chang et al., Nature, 297:7479 (1982); Pulciani, et al., Mol.Cell. Biol., 5:2836 (1985). For example, transfection of normal H-rasDNA has been shown to induce malignant transformation. It is furthernoteworthy that normal ras gene amplification has been observed inseveral human tumors, and has an apparent incidence of about 1%.Pulciani, et al., above; Yokota, et al., Science, 231:261 (1986). Thevarious diagnostic test used to detect ras oncogenes or oncogenic p21have been applied to the detection of normal ras p21 with similarlimited success.

It should be apparent from the foregoing that while there are a numberof diagnostic methods for determining the presence of ras oncogenes, ortheir transforming proteins, there is still a need for fast and reliablediagnostic methods that will permit their routine identification.

By and large, the vast majority of cancer therapeutics function bykilling dividing cells, and because of this lack of specificity, killnormal as well as cancer cells. Thus, despite knowledge of the existenceof normal cellular ras genes, or their oncogenic counterparts, therehave been identified few therapeutics that can interfere with, orreverse the transformed state that are not generally cytotoxic.Valeriote, F. and Putten, L., Cancer Res., 35:2619 (1975). Theexceptions include anti-ras monoclonal antibody, Y13-259, which has beenshown to selectively block the morphologic transformarion induced byoncogenic ras proteins. Mulcahy, et al., Nature, 313:241 (1985). Also,U.S. patent application Ser. No. 938,581 shows a monocional antibodydirected against oncogenic p21 having serine at position 12. Thisantibody, when microinjected into ras transformed cells causes the cellsto revert to a normal cell phenotype. It has no effect on normal cells.Unfortunately, because ras is located on the cytoplasmic side of theplasma membrane, where it interacts with GTP and GAP, large molecularweight molecules such as antibodies may not have immediate therapeuticsignificance in the clinical setting. Thus, a method that willfacilitate the identification of cancer therapeutics is sorely needed.

SUMMARY OF THE INVENTION

In accordance with the present invention, compositions are describedthat are useful both as diagnostics for cancers arising from theexpression of normal cellular or oncogenic ras genes, and in theclinical setting to identify anti-cancer therapeutics effective againstsuch cancers.

A second aspect of the invention relates to compositions consisting ofdefined concentrations of normal cellular or oncogenic ras p21, GAP (oractive fragments derived therefrom), and GTP that are useful in cancerdiagnosis and in clinical tests to identify anti-cancer therapeutics.

A third aspect of the invention consists of methods for purifying GAP,both the intact molecule, and biologically active fragments derivedtherefrom.

A further aspect of the invention is to identify structural regions ofGAP, or fragments derived therefrom, that facilitate use of the moleculein a cancer diagnostic test, or in clinical tests to identifyanti-cancer therapeutics.

A yet further aspect of the invention is a method for curing cancer,whereby anti-cancer therapeutics are identified by their ability tointerfere with GAP binding to a complex consisting of normal oroncogenic ras p21 protein, and GTP; and formulating and administeringthe therapeutics to patients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the TSK phenyl column elution profile and FIG. 1B silverstaining of SDS PAGE fractions thereof.

FIG. 2 shows the SDS gel profile of GAP purified by a three-stepchromatographic scheme consisting of cation, and anion chromatography,followed by a second cation chromatographic step.

DETAILED DESCRIPTION OF THE INVENTION

A better understanding of the invention described herein will berealized by providing a brief description of some of the materials andmethods used in the invention.

The normal cellular ras gene, and its oncogenic counterparts are definedas described by Barbacid, N., Ann. Rev. Biochem., 56:779 (1987).Similarly, the proteins encoded by these genes are also defined asdescribed by Barbacid. Moreover, it will be appreciated that fragmentsof normal cellular p21 that bind GTP, and exhibit GAP stimulated GTPaseactivity are intended to come within the definition of ras p21.

GAP is the acronym for guanine triphosphatase activating protein, and isdefined as a protein having a molecular weight and amino acid sequenceas described herein, and that has the further properties of stimulatingGTPase activity of normal cellular ras p21, while having little or nostimulatory activity when combined with oncogenic ras p21 proteins andGTP. Of course, it will be understood by those skilled in the art thatGAP may also exist as aggregates or multimers under certain conditions,and these forms are intended to come within the scope of the definition.Moreover, the definition is further intended to cover fragments of GAPthat exhibit activity. Exemplary of such a fragment is a molecule havinga reduced subunit molecular weight of about 50,000-60,000 as shownherein.

It will further be appreciated with regard to the chemical structure ofGAP, that its precise structure may depend on a number of factors. Asall proteins contain ionizable amino and carboxyl groups it is, ofcourse, apparent that GAP may be obtained in acid or basic salt form, orin neutral form. It is further apparent, that the primary amino acidsequence may be augmented by derivatization using sugar molecules(glycosylation) or by other chemical derivatizations involving covalentor ionic attachment to GAP with, for example, iipids, phosphate, acetylgroups and the like, often occurring through association withsaccharides. These modifications may occur in vitro or in vivo, thelatter being performed by a host cell through post translationalprocessing systems. It will be understood that such modifications,regardless of how they occur, are intended to come within the definitionof GAP so long as the activity of the protein, as defined herein, is notsignificantly reduced. It is to be further expected, of course, thatsuch modifications to GAP may quantitatively or qualitatively increaseor decrease the biological activity of the molecule, and such chemicalmodifications are also intended to come within the scope of thedefinition of GAP.

As used herein, "chromatography" is defined to include application of asolution containing a mixture of compounds to an adsorbent, or othersupport material which is eluted, usually with a gradient or othersequential eluant. Material elated from the support matrix is designatedeluate. The sequential elation is most routinely performed by isolatingthe support matrix in a column and passing the elating solution(s),which changes affinity for the support matrix, either stepwise orpreferably by a gradient. It will be appreciated that encompassed withinthe definition "chromatography" is positioning the support matrix in afilter and sequentially administering eluant through the filter, or inbatch-mode applications.

The phrase "hydrophobic interaction matrix" is defined to mean anadsorbent that is a hydrophobic solid such as polystyrene resin beads,rubber, silica-coated silica gel, or crosslinked agarose sufficientlysubstituted with hydrophobic functional groups to render the materialhydrophobic. Alkyl substituted agarose and aryl substituted agarose suchas, for example, phenyl or octyl agarose are representative hydrophobicmaterials. Mixtures of materials that are chromatographically separatedon a hydrophobic interaction chromatography matrix are generally firstadsorbed to the matrix in a high salt solution, and subsequentlydesorbed from the matrix by elution in a low salt solution, or ahydrophobic solvent such as a polyol.

"Anion exchange matrix" is defined to mean a solid or gel support matrixthat is charged in aqueous solutions. The support matrix may be agarosesufficiently substituted with amine functional groups to have a netcharge in aqueous solutions. The material to be adsorbed is generallybound to the anion exchange matrix in a low salt solution and isgenerally eluted from the anion exchange matrix in a high salt eluantcontaining anions such as chloride ion which bind to the anion exchangematrix and displace the adsorbed material.

By the phrase "high salt concentration conditions" is meant an aqueoussolution wherein an ionic substance is present to crate conditions ofhigh ionic strength. Ionic strength is defined as is generallyunderstood in the art and can be calculated from the putativeconcentrations of the various ions placed in solution modified by theiractivity coefficient. High salt concentrations that are routinelyemployed are typified by solutions containing high concentrations ofammonium sulfate; however, other salts, such as sodium chloride,potassium chloride, sodium sulfate, sodium nitrate, or sodium phosphatemay also be employed.

The definition of "affinity chromatography" is understood to be similarto that of Wilchek, et al., Methods in Enzymology, 104:3 (1984). In itsbroadest intended definition, "affinity chromatography" is a "method ofpurification based on biological recognition". Briefly, the procedureinvolves coupling a ligand to a solid support, and contacting the ligandwith a solution containing therein a ligand recognition molecule whichbinds to the ligand. Subsequently, the ligand recognition molecule isreleased from the ligand and isolated in pure form. It will beunderstood that a variety of ligands can be employed in affinitychromatography as discussed by Wilchek, et al., and examples of theseinclude lectins, antibodies, receptor-binding proteins and amino acids.

GENERAL DESCRIPTION

The instant invention provides a description of compositions and methodsof using the same for diagnosing cancer, and for identifying cancertherapeutics. The main components of the compositions are ras p21protein, either the normal cellular protein or the oncogenic routants,guanosine triphosphate, or other hydrolyzable guanine nucleotide, andGAP. There are additional reagents present in the compositions, butthese are ancillary to the three main components. Each of the three maincomponents will now be discussed separately.

GAP Purification

Guanosine triphosphatase activating protein, or GAP, is widely expressedin higher eukaryotes. GAP has been detected in cell extracts from humanand mouse normal tissues including brain, liver, placenta, B cells, andplatelets. It has additionally been found in non-transformed cellcultures including NIH 3T3, as well as transformed cell lines, includinghuman mammary cancer cells (MCF-7), retinoblastoma cells CY79), andWilm's tumor (G401). GAP is also present in insect cells such as, forexample, Spodoptera fragipedra. From many of these cells or tissues, GAPmay be isolated, albeit with minor variations in the purificationprotocols and the like.

The general scheme for GAP isolation and purification consist ofreleasing the molecule from the cytoplasm of appropriate cells, tissuesor organs, followed by removing insoluble material and subjecting thesoluble GAP fraction to cation exchange chromatography, followed by asecond chromatographic step wherein the eluant from the cation exchangeris passed over an anion exchanger. GAP is eluted from the anionexchanger, and further purified by subjecting it to a thirdchromatographic step, either hydrophobic chromatography, or a secondcation exchange step.

More specifically, GAP is prepared by releasing the molecule from thecytosol using any number of techniques including freeze thawing,sonication, mild detergent extraction, etc. This procedure is preferablycarried out in a physiologically buffered solution containing one ormore protease inhibitors. Moreover, to further inhibit proteaseactivity, especially those proteases that rely on metal ions foractivation, the extraction solution may contain metal ion chelators. Thepreferred extraction solution is a physiologically balanced saltsolution containing the chelators ethyleneglycoltrichloroacetic acid(EGTA), or ethylenediaminetrichioroacetic acid (EDTA), plus the proteaseinhibitor phenylmethylsulfonylfluoride (PMSF). The metal ionchelator(s), as well as the protease inhibitor(s) are present atconcentrations that effectively inhibit proteolysis, preferably about 5mM and 100 μM, respectively. However, it will, of course, be appreciatedby those skilled in the art that since the types and amounts ofproteases vary depending on the starting material used to extract GAP,that the concentrations that the protease inhibitors or chelators areused at, if indeed used at all, will also vary.

The mixture containing GAP is clarified by centrifugation, or in otherways to remove insoluble material from the aqueous cytosol fraction. Ifthe cytosol fraction contains low amounts of GAP it can be concentratedby any one of several techniques well known to those skilled in the art,including high salt precipitation, such as for example with ammoniumsulfate, or by ultra filtration. If GAP is concentrated byprecipitation, it is preferably subsequently resuspended in a suitablephysiologically balanced salt solution containing protease inhibitor(s)and preferably about 0.1% of a nonionic detergent, such as NP40. Thissolution is then prepared for ion exchange chromatography by dialyzingit against a compatibly buffered chromatographic solution, preferablycontaining millimolar phosphate, a metal ion chelator, a reducing agent,and a protease inhibitor. Additionally, because GAP activity isstimulated by the presence of divalent cations such as magnesiumchloride, it may also be present in the solution. The pH of the solutionis preferably about 6.0.

The GAP dialyzate is then subjected to chromatographic purificationconsisting preferably of three steps. The first involves purificationusing an ion exchange chromatographic step compatible with the GAPextraction buffer. Since the preferred extraction buffer containsphosphate, the initial step is purification of GAP by cation exchangechromatography. The second consists of ion exchange chromatographywherein the ion exchange matrix has the opposite ion binding capacityfrom that of the first ion exchanger employed.

Thus, the preferred purification scheme will consist of applying thephosphate solution containing GAP to a cation exchanger, and eluting GAPtherefrom, preferably using solutions which alter the pH or conductivityof the solution. More preferably GAP will be eluted by applying either agradient or non-gradient salt solution, and most preferably will beeluted using a linear gradient of sodium chloride over the range ofabout 0-0.6 molar.

The preferred cation exchanger is a SP-cellulose cation exchanger. Suchare commercially available from AMF Molecular Separations Division,Meridian, CT. under the brand name ZetaPrep SP cartridges. TheSP-cellulose cation exchanger is an elastic 3-dimensional networkcomposed of cellulosic backbones crosslinked with vinyl polymercontaining pendant sulfopropyl functional groups. The matrix ispreferably adapted for radial flow passage of the GAP solution. The flowrate of the solution through the matrix will depend upon the size andgeometry of the matrix used. It will be apparent to those skilled in theart, however, that care should be taken to avoid exceeding the unitcapacity of the matrix with GAP. If the capacity is exceeded, GAP willnot be totally retained and excess unretained GAP will be present in theeffluent. The capacity of the matrix to retain GAP can be monitored byassaying for GAP in the effluent using one of the assays describedbelow.

Fractious containing GAP are prepared for the second chromatographicstep, that is, anion exchange chromatography. This consists of combiningthe fractions and adjusting the solution to a pH, and ionic strengthcompatible with artion exchange chromatography. A variety of artionexchangers are available, and depending on the type employed, theconcentrations of these reagents will vary. DEAE-Sepharose orTSK-DEAE-5-PW may be employed. The general procedures for preparing andusing these matrices are known to those skilled in the art.

The preferred artion exchanger is TSK-DEAE-5-PW matrix. It is preparedby equilibrating it with a solution containing chloride ions at a pH of8.5. More preferably, the solution will consist of Tfis hydrochloride,pH 8.5 plus a reducing agent, a metal chelator, magnesium chloride, anda protease inhibitor. The concentrations of the metal chelator andprotease inhibitor will vary and depend on how extensively GAP isproteolyzed, and whether the proteases responsible are activated bymetal ions. The concentration of monovalent cations, such as magnesiumchloride and reducing agent can be determined empirically by monitoringGAP activity. Those concentrations which maintain the highest activitywill be utilized. Generally, it is preferred that magnesium chloride andthe reducing agent be present in the range of about 0.5-1 maM, and 0.1-1maM, respectively.

The solution is then passed through the artion exchange matrix whereuponGAP binds to the matrix. GAP is subsequently eluted from the matrixusing solutions which alter the pH or conductivity. The preferredelution method consists of eluting GAP using a linear salt gradientranging from 0-0.6 molar sodium chloride. The purity and activity of GAPso obtained can be monitored by the GTPase assay described below, and bysodium dodecyl sulfate polyacrylamide gel electrophoresis run underreducing conditions. Using these techniques it was determined that GAPhas a molecular weight of about 115,000-120,000 daitons.

The third chromatographic step consist of applying, after the anionexchange chromatography, either a second cation exchange step, or ahydrophobic interaction chromatographic step. The most preferredpurification scheme utilizes a second cation exchange step. Applicationof either of these methods will generally increase the purity of GAP toabout 95%. If a cation exchange column is chosen, the materials andmethods described above are similarly applicable here. Generally, thiswill consist of decreasing the salt concentration present in the anioncolumn eluates and adjusting the pH to about 6.0. Here, as in theinitial cation chromatographic step, several different types of cationexchange matrices can be employed; however, the preferred matrix is aSPTSK column which is run under high pressure.

If hydrophobic chromatography is selected, the ionic strength of theeluate from the artion exchanger should be increased to be compatiblewith hydrophobic interaction chromatography. The solution can then bepassed through a hydrophobic interaction chromatographic matrix, andeluted using techniques known in the art, including decreasing the saltconcentration, or with a chaotropic agent. Either of the lattersolutions may be used alone, or in combination.

A variety of hydrophobic interaction chromatographic matrixes may beutilized. Generally, the materials and methods for utilizing hydrophobicchromatography are described by Shaltie, S., Methods in Enzymology,104:69 (1984). While it is apparent there are many hydrophobicchromatographic materials and methods that may be employed to purifyGAP, phenyl Sepharose is preferred, and it is further preferred that thechromatography be employed under high pressure. The general proceduresfor forming high pressure liquid chromatography involving a phenylderivatized matrix are described by Regmaer, F., Methods in Enzymology,91:137 (1983). The preferred phenyl derivatized matrix is availablecommercially from Bio-Rad Corporation, and is sold under the trade nameBiogel TSK phenyl-5PW.

It will be additionally appreciated by those skilled in the art that analternative purification scheme may conist of a cation and anionchromatographic exchange step, followed by an affinity chromatographicstep. This may be achieved by binding GAP to one or more plant lectinshaving a known carbohydrate specificity compatible with carbohydrateswhich may be present on GAP, or by binding GAP to anti-GAP antibodies.In either event, GAP can then be released from the affinity matrix usingthe appropriate sugar if the matrix is composed of a lectin, or by pH orchaotropic agents if the matrix is composed of antibody.

Because GAP is a protease-sensitive molecule that is broken down intolower molecular weight species having GAP activity, in a preferredembodiment of the invention the entire purification procedure is carriedout rapidly in the cold to reduce protease activity. In general, thistemperature is in a range below 10° C., with a preferred temperaturerange being about 2°-8° C. Most preferred is a temperature of about 4°C.

Finally, it should be noted that while the preferred applications of theion exchange materials described herein is in a column format, it willbe appreciated that they may also be used in batch format as well.

GAP Assay

Several assays have recently been described to measure GAP activity.Trahey, M. and McCormick, F., Science, 238:542 (1987); Adari, et al.,Science, 240:518 (1988). These references are herein incorporated intheir entirety.

GAP may be assayed in vitro, and several different types of in vitroassays can be performed. The preferred assay involves measuring thepresence of GDP resulting from the hydrolysis of GTP. This assayinvolves combining in an appropriate physiologically buffered aqueoussolution empirically determined optimal amounts of normal cellular p21,and α-³² P-GTP, plus GAP. The solution may also contain proteaseinhibitors and a reducing agent. Also, since cations greatly stimulateGAP activity they should be present in an effective amount. Thepreferred cation is magnesium chloride.

The reaction solution is incubated for various times and may beconducted at temperatures typically employed to perform enzymaticassays, preferably 10°-40° C., and more preferably at 37° C. At theappropriate times aliquots are removed and assayed for α-³² P-GDP. Thisis readily accomplished by first separating p21 containing bound α-³²P-GDP from the other reactants in the solution, particularly free α-³²P-GTP. This can be achieved by immunoprecipitating p21 with antibodiesdirected thereto. Immune precipitation techniques and anti-p21antibodies are known, and routinely employed by those skilled in theart. α-³² P-GDT, is released from the immune precipitate preferably bydissolving the sample in a denaturing detergent at an elevatedtemperature, more preferably in 1% sodium dodecyl sulfate at 65° C. forfive minutes, and chromatographing the mixture on a suitable thin layerchromatographic plate. The chromatography is preferably carried out on aPEI cellulose plate in 1M LiCl. α-³² P-GDP is identified by its mobilityrelative to a known standard using suitable radiodetection techniques,preferably autoradiography.

An alternative assay for GAP activity is to substitute γ labeled ³²P-GTP for α-labeled ³² P-GTP in the above assay system, and assay forfree ³² p labeled phosphate using activated charcoal. This assay can becarried out as described by Tjian et al., Cold Spring Harbor Symp.Quant. Biol., 44:103 (1980).

An additional assay does not involve immune precipitation. Rather analiquot from a GAP assay reaction mixture described above can bedirectly subjected to PEI cellulose chromatography in 1M LiCl. Thisassay, however, is most useful for assaying solutions havingsubstantially purified GAP.

Ras p21 Assay

One aspect of the instant invention is that the applicants havediscovered a method whereby normal cellular p21 can be assayed. Althoughother assays present exist, for example, Western blotting using anti-p21antibodies, the instant assay is complementary in nature, andfacilitates the identification and characterization of tumors havingabnormally elevated levels of normal p21. One of the key components inthe assay is GAP, and it may be employed in one or more compositionsutilized in different assay formats. Because GAP specifically stimulatesnormal cellular p21 GTPase activity, GAP can be used to assay for p21 inthe presence of GTPases present in cell extracts.

Tumor cell extracts can be assayed for normal cellular p21 by adding GAPto a solution containing the cell extract, GTP, and the other reagentsdescribed in the preceding section, depending on the degree of GAP orp21 proteolyses and the need for cation stimulation of GAP activity. Ifp21 is present, there will be an enhancement in GTPase activityresulting from GAP stimulation. If p21 is not present, then thestimulation will not be apparent. It is important to note that not onlyis wild-type normal cellular ras p21 assayable by this method, but avariety of non-transforming routants of normal ras p21 can also beassayed. These are shown by Adari, H., et at., above.

It is important to note that because most cells so far studied containGAP, tumor cell extracts will likely contain endogenous GAP that mayreduce or decrease the extent of GTPase stimulation resulting from GAPpresent in the reaction mixture. Thus, it may be desirable, evenpreferable, to partially purity p21 from tumor cell extracts beforerunning the assay. p21 can be purified as described by Trahey, et at.,Mol Cell Biol., 7:541 (1987).

Identification of Anti-Cancer Therapeutics

A second application of the instant compositions described herein is theidentification of anti-cancer therapeutics, particularly those that areeffective against ras related tumors. Without wishing to be held to aparticular theory regarding the role that GAP plays in tumorigenesis,applicants believe that the compositions and methods described below areefficacious in identifying such therapeutics because of an intraeellularcomplex that is associated with tumorigenesis, and that compounds whichprevent the formation of, or which cause its disassociation, can preventtumors, or cause tumor regression. Applicants believe that in the normalcell, cell growth is regulated by down regulation of GTP by p21 suchthat GTP is convened constantly and rapidly to GDP. This conversion isassisted by GAP stimulation of GTP hydrolysis by p21. GAP interactionwith p21 containing bound GTP is believed to be transient, and resultsin the facilitation of normal cell growth. In contrast, tumorigenesis isthought to result from binding of GAP to oncogenic p21 containing boundGTP. In this instance, GAP possibly because of the mutated state of p21,does not readily disassociate from p21/GTP. It is the formation of thiscomplex, GAP/p21/GTP, that is thought to be responsible directly orindirectly for tumor genes. Thus, applicants have developed an assaywhich may be used to identify chemicals that prevent or disrupt theproposed interaction of GAP with p21/GTP, which in turn mimics thenormal cellular p21/GTP complex which apparently does not have GAP boundfor more than very transient times. Consequently, therapeutics whichprevent GAP binding to normal cellular p21, as assayed by a reduction inGTPase activity should have anti-cancer activity.

Accordingly, chemicals can be identified that have anti-cancer activityby performing a GAP assay as described above in the presence of thesuspected anti-cancer therapeutic. If the chemical reduces GAPstimulated GTPase activity, then it is expected to have anticanceractivity.

It will be appreciated that there are to be expected multiple classes ofsuch cancer therapeutics. The two most apparent classes are, fffst,immunoglobulins, or fragments derived therefrom that bind to GAP toeffect GAP interaction with p21/GTP. The second class consists of smallmolecular weight compounds that bind to either p21 or GAP at theirinteractive sites. Thus, it will be appreciated that molecules whichbind either to GAP, or to p21 are assayable by a reduction of GAPstimulated GTPase activity.

An additional class of cancer therapeutics which may be identified bythe instant assay are those that bind to p21 at a site remote from whereGAP and p21 interact which induces n conformational change in p21,thereby altering GAP binding to the molecule.

Having generally described the invention, examples of particularapplications of the invention will be presented below. However, it willbe understood by those skilled in the art that the examples arepresented in the spirit of illustration only, and that they are notintended to limit the scope of the invention.

EXAMPLE I Purification of GAP-Cation/Anion/Hydrophobic Chromatography

GAP was isolated from 300 g of human placentas by the followingthree-step chromatographic procedure. Placentas were obtained shortlyafter delivery, and kept on ice until they were processed. After it wasdetermined by standard tests that the placentas were free of HIVantibodies, they were processed as follows. The initial step consistedof mechanically removing connective tissue, and ridding the placentas ofexcess blood by multiple soakings in phosphate buffered saline (PBS).The placentas were then fragmented by freezing the tissue at -70° C.,followed by placing the tissue in solution of PBS containing 5 mM EGTA,100 μM PMSF and disrupting the tissue in a blender until a uniform, finesuspension was apparent. The suspension was centrifuged at 100,000×g toremove insoluble debris, the supernatant removed and the proteinaceousmaterial therein precipitated with 40% ammonium sulfate. The ammoniumsulfate was removed, and the precipitated proteins resuspended in PBScontaining 0.1% NP40 and 100 μM PMSF. This solution was immediatelydialyzed against 20 mM potassium phosphate, 1 mM MgCl₂, 5 mM EGTA, 0.1mM DTT, 100 μM PMSF, pH 6.1 for six hours. This solution was thenimmediately chromatographed on a cation matrix, S-Sepharose (fast flow,obtainable from Pharmacia Corporation), preequilibrated in 20 mMpotassium phosphate, 1 mM MgCl₂, 5 mM EGTA, 0.1 mM DTT, 100 μM PMSF, pH6.1.

Proteins absorbed to the cation exchanger were elated with a linear saltgradient containing 0-0.6M sodium chloride. Using the GAP assaydescribed below, most of the GAP activity was shown to be present in twopeaks, a major peak elating at a sodium chloride concentration of100-150 mM, and a minor peak eluting at a sodium chloride concentrationof 220-300 maM. The major peak was dialyzed against 30 mM Tris-HCl, 1 mMmagnesium chloride, 1 mM EGTA, 0.1 mM DTT, 100 μM PMSF, pH 8.5. Thedialyzate was applied to a anion exchange column, TSK-DEAE-5-PW(150×21.5 mm). The amon exchange matrix was treated with a linear saltgradient ranging from 0-0.6M sodium chloride to elute the adherentproteins. Most of the GAP activity elated at a sodium chlorideconcentration of about 130 mM NaCl. Those fractions containing GAPactivity were pooled, brought to 0.5M ammonium sulfate, and passedthrough a hydrophobic column, phenyl-TSK HPLC. Proteins were elated fromthe hydrophobic column using a crisscross gradient consisting ofincreasing ethylene glycol 0-30%, and decreasing ammonium sulfate,0.5-0M. The majority of GAP activity eluted at a concentration of 24%ethylene glycol and 0.1 molar ammonium sulfate. GAP activity assays, asperformed below, correlated with a protein band of about 120,000daltons, as revealed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis on 6% gels run under reducing conditions (FIGS. 1A and1B).

EXAMPLE II Purification of GAP-Cation/Anion/Cation Chromatography

A second preferred procedure was employed to purify GAP. Human placentaswere again obtained shortly after delivery, and soaked in ice cold PBS,and homogenized and clarified as described in Example I. Ammoniumsulfate was again added to the clarified homogenate to a finalconcentration of 40% to precipitate proteinaceous material. The ammoniumsulfate solution was allowed to stand for one hour at 4° C. prior torecovering the precipitated proteinaceous material by centrifugation for15 minutes at 10,000×g. The pellet was resuspended in PBS containing0.1% NP40 and 100 μM PMSF. This solution was dialyzed for six hours at4° C. against 20 mM potassium phosphate, pH 6.1, containing 1 mM MgCl₂,5 mM EGTA, 0.1 mM DTT, and 100 μM PMSF. Because GAP is susceptible toproteolysis, longer dialysis times are not desirable.

The GAP dialyzate was diluted three-fold with 4 mM potassium phosphate,pH 6.1, containing 0.02M MgCl₂, 1 mM EGTA, 0.1 mM DTT, and 100 μM PMSFto lower the conductivity of the solution to 1 millisiemens. Thisconductivity is compatible with application of the dialysate to aS-Sepharose cation exchange column. The dialysate was clarified bycentrifugation at 10,000×g for 10 minutes, followed by a furtherclarification step consisting of filtration through a 0.45 μM filter,prior to adding the dialysate to the S-Sepharose column (fast-flow,Pharmacia). Most of the contaminating proteins passed through theS-Sepharose column, and the adsorbed proteins eluted with a 1.5 litersalt gradient consisting of 0-0.6M NaCl. Those fractions containing GAPactivity were identified using the GAP assay described below.

As observed in the first example, GAP eluted from the cation exchangecolumn in predominantly two major peaks. The first peak eluting over asodium chloride concentration of 100-150 mM was pooled and dialyzedagainst 30 mM Tris-HCl buffer, pH 8.5, containing 1 mM EGTA, 1 mM MgCl₂,0.1 mM DTT and 100 μM PMSF. The solution was dialyzed at 4° C., andclarified by filtration with a 0.45 μM filter. The filtrate was dividedinto equal halves, and each half purified using two consecutive artionexchange columns.

The two tiltrates were separately loaded onto a TSK-DEAE-5-EW columnhaving the dimensions 150×21.5 mm. The column was preequilibrated in theTris-hydrochloride, pH 8.5 dialysis buffer described above. GAP waseluted from the column with a 60-minute 0-0.6M NaCl gradient with a flowrate of 3 ml/minute. The majority of the GAP activity from bothflitrates eluted as a single peak at a sodium chloride concentration ofabout 130 raM. Sodium dodecyl sulfate, polyacrylamide gelelectrophoretic analysis of the DEAE fractions showed that GAP was themajor protein in the peak activity fractions. Fractions containing GAPfrom both purifications were pooled and diluted 5-fold into 2 mMpotassium phosphate, pH 6.1, containing 0.1 mM EGTA, 10 μM DTT, 10 μMPMSF to lower the salt concentration to insure that the solution waschromatographically compatible with a second cation exchangechromatographic step, that is, chromatographed over a SP-TFK column. ThepH of the solution was checked and adjusted to pH 6.1 with sodiumacetate if necessary 3 M pH 4.8. Both of the GAP fractions isolated fromthe DEAE columns were further purified separately over a cation column,TSK-SP-5-PW having dimensions of 75×7.5 min. A solution containing 20 mMpotassium phosphate, pH 6.1, containing 1 mM EGTA, 0.1 DTT, and 0.1 mMPMSF was passed through the column, followed by eluting GAP with a45-minute, 0-0.6M sodium chloride gradient at 1 ml per minute. Thosefractions containing GAP were identified using the assay described belowand sodium dodecyl sulfate polyacrylamide gel electrophoresis. GAPactivity corresponded to a protein having a molecular weight of about116,000 daltons. Amino acid analysis was performed on the purified GAPto determine protein concentration. Starting with about 300 grams ofhuman placenta, approximately 430 micrograms of purified GAP wasobtained. FIG. 2 shows the SDS PAGE analysis of GAP at the variousstages of purification described above.

EXAMPLE III GAP Amino Acid Sequence

The protein having a molecular weight of 120,000 obtained by thepurification method of Example I was electro-eluted from a 6% sodiumdodecyl sulfnte, polyacrylamide gel in 0.05 molar ammonia bicarbonatecontaining 0.1% sodium dodecyl sulfate. The procedure followed isdescribed by Hunkapillar et al. The electroeluted protein was fragmentedfor internal sequencing using lysyl endopeptidase (5% w/w, 18 hours at40° C., WAKO). Peptides were fractionated by reverse-phase highperformance liquid chromatography using a Brownlee Aquapore RP-300cartridge (100×2.1 mm, Applied Biosystems). Peptides were eluted with anAcetonitrile gradient from 0-70% in 120 minutes (Buffer A, 0.1%trifluoroacetic acid (TFA) in H20; Buffer B, 0.085% TFA in 85%acetonitrile). Automated sequence analysis of the peptides was conductedon an Applied Biosystems 470A gas-phase sequencer as reported). Apeptide characteristic of GAP has the following amino acid sequence: I MP E E E Y S E F K.

EXAMPLE IV GAP Assay

Approximately 0.8 micrograms of H-ras protein obtained as described byTrahey, et al., supra was bound to α-³² P-(GTP followed by precipitationof the complex with 13 micrograms of an anti-ras antibody, 157-181, thatrecognizes the carboxyl terminal end of the molecule. SpecificAlly,157-181 recognizes the carboxyl terminal residues at positions 157-181.Adari, et at., Science, 280:518 (1988). Next, 10 micrograms ofsheep-antimouse IgG, and 10 microliters of protein A-Sepharose beadswere added. As a control, the same reactants were combined except thatrat IgG replaced 157-181, and goat anti-rat IgG replaced sheepanti-mouse IgG. The pellets were washed with 20 mM tris hydrochloride,pH 7.4, containing 20 mM sodium chloride, 1 mM magnesium chloride and 1mM DTT and resuspended in the same solution. Four microliter aliquots ofthe immune complex were then mixed with 10 microliters of GAP, or, as acontrol, buffer without GAP. After 60 minutes incubation at roomtemperature the Sepharose beads were washed again, and the boundnucleotides analyzed using thin layer chromatography with 1M LiCl as thesolvent. The thin layer plate was audioradiographed for one-two hoursafter which it was developed. The audioradiograph revealed that additionof sufficient GAP causes the near complete hydrolysis of GTP to GDP,whereas very little GTP hydrolysis occurs in the control lacking GAP.The assay detects GAP in a semi-quantitative, dose-dependent fashion.Quantitation can be improved by scraping the relevant regions of theplate and measuring cpm in GDP by use of a gamma counter. The immuneprecipitation controls having rat IgG substituted for the mouseantibodies revealed no GTP or GDP.

In addition to the above method, GAP can be preferably assayed asfollows. Four μM normal cellular p21 was dissolved in a buffercontaining 80 mM β-glycerophosphate, 5 mM MgCl₂, 1 mM DTT, pH 7.5, plus255 μM [α-³² P] GTP (16 Ci/mmol), 4 mM ATP, and bovine serum albumin(2.5 mg/ml). The mixture was preincubated for 30 minutes at 37° C.,followed by the addition of either a sample suspected of contained GAP,or an equal volume of buffer. After one hour at room temperature themonoclonal antibody Y13-259 in the presence of 0.5% NP40 was added in anamount sufficient to bind all the p21 present in the solution. Next,goat anti-Rat Ig-Protein A Sepharose was added to collect p21 bound toY13-259, and the immune complex isolated and washed ten times in 20 mMTris-HCl, pH 8.0, 1130 mM NaCl, 5 mM MgCl₂, and 0.5% NP40. To determinethe extent of GTP binding and hydrolysis during these steps a controlwas run consisting of adding 5 μg of p21 immediately before addingY13-259.

Nucleotides were eluted from p21 with 1% SDS, 20 mM EDTA at 65° C. forfive minutes and chromatographed on PEI Cellulose in 1M LiCl. GTP andGDP were visualized using standard autoradiographic techniques. Theresults showed that normal cellular p21 affects a nearly completeconversion of GTP to GDP when compared to mutant ras oncogenic proteinsAsp 12 and Vat 12 assayed similarly. Moreover, little or no GTP or GDPwas detected in the control sample.

The assays described above are presented in more detail by Trahey andMcCormick in Science, 238:542 (1987), and by Adari et al. in Science,240:518 (1988). Both of these references are hereby incorporated byreference.

EXAMPLE V Identification of Normal Cellular ras 21 in Tumor Samples

A variety of technical procedures can be employed using GAP to assay forthe expression of normal cellular p21 in tumor tissues. One proceduremay consist of isolating p21 by extraction techniques well known tothose skilled in the art, Trahey et al. Molecular and Cellular Biol.7:541 (1987), and combining the extract with a solid support matrix,preferably agarose beads that have bound anti-p21 antibodies.Alternatively, p21 may be assayed directly without purification. Theantibodies should be selected so as to bind normal cellular p21 withoutaffecting its intrinsic GTPase activity. Such antibodies as well asmethods whereby they are bound to a solid support, are well known tothose skilled in the art. Thus, p21 present in the tumor extract can bebound to anti-p21 antibody, which in turn is bound to agarose beads. Thebeads are then washed with a buffer compatible with performing theGTPase assay described in Example III, followed by the addition of anappropriate amount of α³² -GTP, GAP, and the other reagents described inExample III to optimize the GTPase stimulation by GAP. This mixture isincubated at 37° C., and GTP hydrolysis measured by thin layerchromatography using 1 molar lithium chloride as the solvent.

Several controls should be run to insure that GAP-stimulated GTPaseactivity is indeed due to the presence of normal cellular p21. Thesecontrols are readily apparent to those skilled in the art, and includerunning in parallel in the assay n reaction mixture lacking normalcellular p21, as well as replacing the anti-p21 antibodies withantibodies that lack p21 specificity. Additional, it will be appreciatedthat the amount of normal cellular p21 in the tumor can be quantitatedby running known amounts of normal cellular p21 in the assay, andconstructing a standard curve wherein the increase in GAP stimulatedGTPase activity is related to the amount of normal cellular p21.

It is worth noting that the contribution of endogenous GAP present inthe tumor tissue extract to GAP stimulated GTPase activity measured nthe assay is minimized by both purifying p21, as well as binding p21 tobeads that are readily washed to remove any residual contaminatingendogenous GAP prior to performing the assay.

EXAMPLE VI Identification of Cancer Therapeutics

Molecules having anti-cancer activity can be identified by performingthe GAP-stimulated GTPase assay as described in Example III in thepresence of cormpounds suspected of having such activity. A reduction inGAP stimulated GTPase activity indicates that the compounds should haveanti-tumor activity. It will be appreciated that water soluble compoundscan be added directly to the assay mixture, whereas non-water solublecompounds may be dissolved in art organic solvent, and then added to thereaction mixture. The mount of organic solvent should not substantiallyinterfere with the enzymatic reaction.

Having generally described the invention, it will be understood by thoseskilled in the art that there exists a wide range of equivalentmaterials and methods that can be substituted for those shown hereinwithout effecting the spirit or scope of the invention. The scope of theinvention should not be construed as being limited other than by theappended claims.

What is claimed is:
 1. A isolated and substantially pure proteinmolecule comprising art apparent molecular weight of about115,000-120,000 daltons as assessed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis run under reducing condition thatsimulate non-oncogenic ras p21 GTPase activity but does notsubstantially affect GTPase activity of oncogenic routants.
 2. A methodof purifying a molecule having art apparent reduced molecular weight ofabout 115,000-120,000 daltons that simulates non-oncogenic ras p21GTPase activity, but does not substantially affect GTPase activity ofoncogenic routants, from a solution containing the same, comprising thesteps of:contacting said solution with cation exchange chromatographicmaterial for a time sufficient for said molecule to bind to saidmaterial; forming a first eluate containing said molecule by elutingsaid molecule from said cation chromatographic material by contactingsaid chromatographic material with an aqueous salt solution; identifyingfractions in said first eluate having said molecular, and reducing thesalt concentration present in said fractions to be compatible with anionexchange chromatography; forming a second eluate by contacting saidfractions of said first eluate containing said molecule with artionexchange chromatographic material for a time sufficient for saidmolecule to bind to said material, and eluting said molecule from saidmaterial artion exchange material with art aqueous salt solution;forming a third eluate by contacting said second eluate with a secondcation exchange chromatographic material for a time sufficient for saidmolecule to bind to said material, and eluting said material from saidsecond cation exchange chromatographic material; and identifyingfractions of said third eluate containing said molecule.
 3. The methodas described in claim 1 wherein said molecule is purified from humanplacenta.
 4. The method as described in claim 1 wherein saidpurification is conducted in solutions containing one or more proteaseinhibitors at concentrations that effectively inhibit proteolysis ofsaid molecule.
 5. The method as described in claim 4 wherein saidmolecule is present in a solution comprising a reducing agent in anamount effective to preserve the activity of said molecule.
 6. Themethod as described in claim 5 wherein said purification is conducted insolutions containing one or more metal ion chelators at concentrationsthat prevent substantial loss of activity of said molecule.
 7. Themethod as described in claim 6 wherein said purification is conducted insolutions containing cations in an amount that substantially maintainsthe activity of said molecule.
 8. The method as described in claim 7wherein said cation is magnesium chloride.
 9. The method as described inclaim 1 wherein said solution contains about 1 mM magnesium chloride, 5mM EGTA, 0.1 mM DTT, and 100 μM PMSF.
 10. The method as described inclaim 1 wherein said tirst cation exchange chromatographic material isS-Sepharose.
 11. The method as described in claim 1 wherein said anionexchange chromatographic material is diethyl aminoethyl.
 12. The methodas described in claim 1 wherein said second cation chromatographicmaterial comprises sulfopropyl functional groups.
 13. The method asdescribed in claim 10 wherein said second cation exchange material isTSK-SE-5-PW.
 14. The method as described in claim 1 wherein ahydrophobic interaction chromatographic material is substituted for saidsecond cation exchange material, and said second eluate from said anionexchange chromatographic material is made compatible with saidhydrophobic interaction chromatographic material by increasing the ionicstrength of said second eluate, and forming a third eluate by elutingsaid molecule from said hydrophobic interaction chromatographicmaterial.
 15. The method as described in claim 14 wherein said moleculeis eluted from said hydrophobic interaction chromatographic materialwith an aqueous low salt solution, or an effective mount of a polyol.16. The method as described in claim 14 wherein said hydrophobicinteraction chromatographic material is under high pressure.
 17. Anisolated and substantially pure protein molecule as described in claim1, wherein said non-oncogenie ras p21 GTPase activity is associated withH-ras.
 18. An isolated and substantially pure protein molecule asdescribed in claim 1, wherein said non-oncogenic ras p21 GTPase activityis associated with N-ras.